Cancer is a somatic genetic disease that strikes over one million Americans per year. The majority of tumors arise due to somatic mutations in a small set of known oncogenes and tumor suppressor genes. Somatic mutations can occur anywhere in the gene, but the specific location and identity of the mutations greatly influences tumor aggressiveness and response to treatment. However, these mutations are difficult to characterize using existing technologies because DNA extracted directly from the tumor comes from a mixture of cancerous and non-cancerous tissue. The goal of this project is to develop Peptide Mass Signature Genotyping (PMSG) into a low-cost and reliable method for identifying and characterizing oncogene and tumor suppressor mutations in heterogeneous DNA samples taken directly from tumors. The initial test will analyze the TP53 gene, the most commonly mutated gene in human cancers. PMSG employs protein expression systems to generate recombinant, tagged peptides from more than one reading frame of the DNA under test after which the peptides are purified and analyzed using MALDI-TOF mass spectrometry and interpretive software. The large bandwidth of the MALDI mass spectrum permits multiplexed analysis of numbers of peptides simultaneously, each of which represents several hundred bases of DNA sequence. However, the analysis is limited to a significant degree by the presence of stop codons in some reading frames of the target sequence and so, protein expression capability needs to be expanded by utilizing suppressor tRNA mutations in the bacterial expression system in combination with serial affinity capture. This will result in a test that covers all of the coding exons of the gene and their associated splice sites. The most labor intensive steps in the process will be automated to achieve a standardized, higher throughput, lower cost analytical test. Cancer-related genes will be the initial focus, but the automated platform will find commercial application in a number of additional fields where it is useful or necessary to scan mixed populations of DNA for mutations. These include infectious disease, inherited disease, and pharmacogenetics.